Although the concept of VH replacement has a lengthy history, the natural occurrence and molecular basis of VH replacement in human B cells have just been realized from our recent studies. VH replacement occurs through RAG-mediated secondary recombination involving the cryptic RSS (cRSS) within the rearranged VHDJH region and the 23 bp RSS from an upstream VH gene. VH replacement occurs in bone marrow immature B cells during human B cell development and contributes to about 5% of the periphery B cell repertoire in humans. Our recent studies showed that the frequencies of VH replacement products are significantly elevated in autoimmune diseases and in IgH genes encoding anti-viral antibodies, suggesting an unrealized important function of VH replacement. The current proposal focuses on the molecular regulation of VH replacement in human immature B cells. We found that crosslinking BCR strongly induces VH replacement in the EU12 <HC+ cells and in primary immature B cells. It has been shown that activation of NF-kB is required for the induction of RAG gene expression and Ig: editing in murine immature B cells. We found that crosslinking BCR induces NF-kB DNA binding activities and blocking NF-kB activation with CAPE inhibits VH replacement in the EU12 <HC+ cells. Based on these observations, we hypothesize that VH replacement is regulated by BCR-mediated signaling in human immature B cells and might be further enhanced by elevated levels of inflammatory cytokines or costimulatory molecules in autoimmune diseases. (1) We will use real time LM-PCR method to determine the relative rate and occurrence stage of VH replacement in comparison with Ig: gene editing in immature B cells from healthy donors and rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) patients. (2) We will modulate BCR signaling by knocking-down BLNK expression using specific siRNA and by activating or blocking NF-kB activities to determine the responsible signaling events for inducing VH replacement. (3) We will analyze the accessibilities to VH genes in different B cell subsets by detection of germline transcription, histone acetylation, and by RAG-mediated in vitro cleavage assays to determine if VH replacement is controlled through regulating the IgH locus accessibility; we will also determine if the accessibilities to upstream VH genes are regulated by BCR or co-stimulatory signaling in normal immature B cells and if these accessibilities are abnormally regulated in RA and SLE immature B cells. Understanding the molecular regulation of VH replacement has profound clinical implications, because VH replacement products are highly enriched in autoimmune diseases and anti-viral responses. PUBLIC HEALTH RELEVANCE STATEMENT: VH replacement occurs through RAG-mediated secondary recombination involving the cryptic recombination signal sequence (cRSS) within the rearranged VH gene and a 23 bp RSS of an upstream VH gene. Although the concept of VH replacement has a length history, the molecular basis and natural occurrence of VH replacement in human B lineage cells have just been realized from our recent studies. Normally, VH replacement contributes to about 5% of the IgH repertoire in the periphery B cells of healthy donors. Strikingly, the frequencies of VH replacement products are significantly elevated in IgH genes derived from different autoimmune diseases and in IgH genes encoding various anti-viral antibodies. The current proposal focuses on the molecular regulation of VH replacement in human immature B cells. We found that crosslinking B cell antigen receptor (BCR) strongly induces VH replacement in human immature B cells. Understanding the molecular regulation of VH replacement will have important clinical implications, because the frequencies of VH replacement products are significantly elevated in autoimmune diseases and anti-viral responses.